Sphingosine analogues

ABSTRACT

The present invention aims to provide a novel sphingosine analogue, which is useful as an intermediate for syntheses of novel lipid derivatives such as sphingolipid derivatives and the like that can regulate the effects of sphingolipid. 
     The present invention relates to a sphingosine analogue represented by the general formula (I) described below.                    
     In the formula, as for Q 1 , Q 2  and Q 3 , Q 1  and Q 2 , which are the same or different each other, are hydrogen, alkyl groups having 1-4 of carbon atoms, acyl groups having 2-5 of carbon atoms, or protecting groups of the amino group, and Q 3  is a hydrogen or a protecting group of the hydroxyl group; or Q 2  and Q 3  make up an isopropylidene group and Q 1  is a hydrogen or a protecting group of the amino group. Q 4  and Q 5 , which are the same or different each other, are hydroxyl groups, acyl groups having 2-5 of carbon atoms, —O—Q 6 , or hydrogen; or Q 4  and Q 5  make up a covalent bond. Q 6  is a protecting group of the hydroxyl group. X 1  is —COOH, —CONH 2 , —CO—Q 7 , —CH 2 OH, or —CH 2 O—Q 8 . Q 7  is a protecting group of the carboxyl group, and Q 8  is a protecting group of the hydroxyl group.

This application is a continuation of PCT/JP98/01038 Mar. 12, 1998.

TECHNICAL FIELD

The present invention relates to a novel sphingosine analogue, which isuseful as an intermediate for syntheses of TKR1785's and theirderivatives useful as drugs for the treatment of fungal infections,allergic diseases, etc., and also as an intermediate for syntheses ofnovel sphingolipid derivatives. The present invention also relates to aprocess for production of a novel sphingolipid.

PRIOR ART

Sphingosine is a compound having the chemical formula shown in thegeneral formula described below, in which Y¹ is hydrogen. It is knownthat various sphingolipids having sphingosine as a constituent arewidely distributed in the living body including on the surface of cellmembranes of cells in the nervous system. Also we knowglycosphingolipids binding one or several kinds of sugars as Y¹ via aglycoside bond to a ceramide having a fatty acid bound to the aminogroup of sphingosine via a peptide bond, and sphingophospholipids,including sphingomyelin, binding a phosphoric acid and a base such ascholine or ethanolamine as Y¹ to the above-mentioned ceramide.

A sphingolipid is one of the lipids having important roles in the livingbody. We know a disease called lipidosis which is caused by accumulationof a specified sphingolipid in the body concomitant with theabnormalities in the metabolic pathways due to respiration deficiencyand others. Attractive effects of sphingolipids present on the cellmembranes include functions in the regulation of cell growth anddiscrimination of each cells; functions in the developments anddifferentiation; functions in nerves; involvement in the infections andmalignancy of cells; and others. Lots of physiological roles of sucheffects remain to be solved. Recently a possibility that ceramide, aderivative of sphingosine, has an important role in the mechanism ofcell signal transduction is indicated, and studies about its effects onapoptosis and cell cycle have been actively performed.

Fungi and plants have sphingolipids and the major sphingosine containedin these organisms has the formula described below. It is known thatthese lipids have important roles in the cell growth of fungi andplants, but details of the roles remain to be solved.

Recently it has been known that derivatives of sphingolipids and theirrelated compounds exhibit a variety of biological activities throughinhibition or activation of the metabolism pathways. These compoundsinclude inhibitors of protein kinase C, inducers of apoptosis,immuno-suppressive compounds, antifungal compounds, and the like.Substances having these biological activities are expected to be usefulcompounds for various diseases.

ABSTRACT OF THE INVENTION

The present invention intends to give a novel sphingosine analogue thatis useful as an intermediate for synthesis of a novel lipid derivative,such as sphingolipid derivatives and the like, capable of controllingthe function of sphingolipid.

In the course of a search for novel biologically active compounds, theinventors isolated lots of microorganisms, obtained biologically activecompounds produced by the microorganisms, and studied on the biologicalproperties. We discovered novel biologically active compounds TKR1785's,which were active against pathogenic fungi including Candida,Aspergillus, Cryptococcus and Malassezia in the culture broth of astrain belonging to Penicillium sp. The inventors also found that theTKR1785's inhibit enzymes involved in allergic reactions.

In this specification, TKR1785's are compounds shown in the followinggeneral formula (II). TKR1785's described above include TKR1785-I shownas the following formula (IIa) and TKR1785-II (IIb).

The inventors succeeded in preparation of novel sphingosine analoguesrepresented by the following formula (III) by hydrolysis of TKR1785'sdescribed above. Furthermore they revealed that the compounds are usefulintermediates for synthesis of TKR1785's.

Also, the inventors found that the group of compounds described in thefollowing general formula (I), which includes the compound described inthe above formula (III) as the representative, is useful to synthesize anovel biologically active compound including novel sphingolipidanalogues or others, and then accomplished the present invention.

In the formula, as for Q¹, Q² and Q³, Q¹ and Q², which are the same ordifferent each other, are hydrogen, alkyl groups having 1-4 of carbonatoms, acyl groups having 2-5 of carbon atoms, or protecting groups ofthe amino group, and Q³ is a hydrogen or a protecting group of thehydroxyl group; or Q² and Q³ make up an isopropylidene group and Q¹ is ahydrogen or a protecting group of the amino group. Q⁴ and Q⁵, which arethe same or different each other, are hydroxyl groups, acyl groupshaving 2-5 of carbon atoms, —O—Q⁶, or hydrogen; or Q⁴ and Q⁵ make up acovalent bond. Q⁶ is a protecting group of the hydroxyl group. X¹ is—COOH, —CONH₂, —CO—Q⁷, —CH₂OH, or —CH₂O—Q⁸. Q⁷ is a protecting group ofthe carboxyl group, and Q⁸ is a protecting group of the hydroxyl group.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an ultraviolet absorption spectrum of the bioactive substanceTKR1785-I. The ordinate represents wavelength (nm) and the abscissarepresents absorbance.

FIG. 2 is an infrared absorption spectrum of the biologically activecompound TKR1785-I. The ordinate represents transmittance (%) and theabscissa represents wave number (cm⁻¹).

FIG. 3 is an ¹H-NMR spectrum of the biologically active compoundTKR1785-I. The ordinate represents the intensity of signal and theabscissa represents chemical shift (ppm).

FIG. 4 is a ¹³C-NMR spectrum of the biologically active compoundTKR1785-I. The ordinate represents the intensity of signal and theabscissa represents chemical shift (ppm).

FIG. 5 shows an HPLC elution pattern of the biologically active compoundTKR1785-I. The ordinate represents retention time (min.) and theabscissa represents the relative intensity of ultraviolet absorption.

FIG. 6 is a ¹H-NMR spectrum of the biologically active compoundTKR1785-II. The ordinate represents the intensity of signal and theabscissa represents chemical shift (ppm).

FIG. 7 is a ¹³C-NMR spectrum of the biologically active compoundTKR1785-II. The ordinate represents the intensity of signal and theabscissa represents chemical shift (ppm).

FIG. 8 shows an HPLC elution pattern of the biologically active compoundTKR1785-II. The ordinate represents retention time (min.) and theabscissa represents the relative intensity of ultraviolet absorption.

DETAILED DESCRIPTION OF THE INVENTION

Following is the present invention now described in detail.

Sphingosine analogues of the present invention are represented by thegeneral formula (I) described above. The above alkyl group having 1-4 ofcarbon atoms is not particularly restricted, but includes, for example,methyl, ethyl, propyl, i-propyl, n-butyl, t-butyl group, and the like.

The above acyl group having 2-5 of carbon atoms is not particularlyrestricted, and includes, for example, acetyl, propionyl, butyryl,valeryl group, and the like.

The protecting group of the above amino group is not particularlyrestricted, but includes, for example, t-butoxycarbonyl (Boc),trichloroethoxycarbonyl (Troc) group, and the like.

The protecting group of the above hydroxyl group is not particularlyrestricted, but includes, for example, benzyl (Bzl), acetyl, methyl,trimethylsilyl group, and the like.

Q⁷ described above is a protecting group of the carboxyl group. Theprotecting group of the above carboxyl group is not particularlyrestricted, but includes, for example, a phenacyl (Pac) group, Bzlgroup, and the like.

Q⁶ and Q⁸ described above are protecting groups of the hydroxyl group.The protecting group of the above hydroxyl group is not particularlyrestricted, but includes, for example, those described above.

Sphingosine analogues described above are included in a compoundrepresented by the general formula (I) described above and include, forexample, the compounds shown in Table I described below.

The compound represented by the above formula (III), which is shown inTable 1 as the compound (1), can be converted to an acyl derivative ofthe hydroxyl or amino group by means of a conventional method using acidanhydride, acid chloride, or the like. The compound represented by theabove formula (III) can be converted to an N-alkylated derivative of theamino group by using sodium hydride and alkyl iodide. In addition, thecompound represented by the above formula (III) can be converted to anamide by methylation of the carboxyl group followed by ammonolysis, orto an alcohol by reduction using lithium alminium hydride (LiAlH₄),sodium borohydride (NaBH₄), or the like.

The compound represented by the above formula (III) can be converted toa compound shown by the general formula (I) except for the compoundshown by the above formula (III) by means of a selective modification ofa specified functional group selected from the hydroxyl, amino, orcarboxyl group, which the compound represented by the above formula(III) has, through selective reaction or use of an appropriateprotecting group. In this case, various protecting groups which areoften used for the peptide synthesis can suitably be available. Suchprotecting groups include protecting groups of the amino group,protecting groups of the carboxyl group, or protecting groups of thehydroxyl group described above.

The protecting group described above is respectively removed in need bya corresponding known elimination reaction or its applied eliminationreaction, and an objective compound is obtained. In the case that aprotecting group that can be eliminated by a different condition isused, the elimination reaction carried out as described above can makeeasily a selective modification. For example, a selective modificationof the amino group can be carried out by protection of the amino groupby a Boc group, protection of the hydroxyl group by an acetyl group,protection of the carboxyl group by a methyl group, standing at anacidic condition to remove and eliminate the Boc group selectively,formation of a peptide bond with an organic acid, and alkali hydrolysisthereafter; and a compound in which only the amino group is modifiedselectively is obtained. If a compound having a hydroxyl group insteadof the carboxyl group is used as a starting material and the hydroxylgroup is protected by acyl group, a ceramide analogous compound can beobtained.

Among the compounds represented by the above general formula (I) of thepresent invention, the compound represented by the above formula (III)can be produced by hydrolysis of TKR1785's represented by the abovegeneral formula (II) such as TKR1785-I or TKR1785-II. For example, thecomposition can be obtained by treatment of TKR1785-I shown as the aboveformula (IIa) with acid hydrolysis, e.g. carrying out the decompositionunder the condition of 6N HCl, at 110° C. for over night or the likewhich is used for the hydrolysis of the peptide bond, followed byneutralization of the reaction solution and being adjusted to alkali.The compound prepared is isolated by neutralization of the reactionsolution again, extraction with an organic solvent such as chloroform, amixture of chloroform/methanol or the like, and if necessary, furtherpurification using an absorption chromatography using silica gel or areversed phase partition chromatography using a chemical-bonded silicagel. The lactone represented by the below formula (IV) can be obtainedwhen TKR1785-I is subjected to acid hydrolysis under the conditionsimilar to that described above, concentration and purification. Forexample, this compound is available to introduce Boc groups to the aminogroup alone or both of the amino and hydroxyl groups by Bocderivatization. Alkali hydrolysis thereafter leads to synthesize onecompound represented by the above general formula (I).

Additionally, as to a compound in which Q⁴ and Q⁵ of the above generalformula (I) are bound, including a compound represented by theabove-mentioned formula (III), a compound having a double bond betweencarbon 5 and carbon 6, such as the compound (10) shown in Table 1described below, can be easily obtained from the lactone shown above asthe formula (IV) through dehydration by treatment with sulfuric acid orthe reaction with thionyl chloride in pyridine, and following alkalihydrolysis. The compound having a hydroxyl group as Q⁴ of the abovegeneral formula (I) can be obtained by direct introduction of a hydroxylgroup to the obtained dehydration product of the above formula (IV)treating with concentrated sulfuric acid, or by alkali hydrolysis afterits epoxidation by reduction.

TKR1785's described above, thus TKR1785-I and TKR1785-II, are preparedby culturing a strain producing TKR1785's and belonging to Penicilliumsp., and isolating them from the cultured broth thereafter. The strainuseful to produce TKR1785's described above is exemplified byPenicillium sp. TKR1785 (referred as “strain TKR1785” thereafter). Thatis, strain TKR1785 is inoculated into a nutrient medium and cultured inliquid to obtain TKR1785's described above.

The culture described above is carried out at 15 to 25° C. preferably,and the incubation for 3 to 11 days usually gives a sufficientproduction. TKR1785's accumulated in the cultured product are obtainedby purification utilizing their physicochemical and biologicalproperties. The purification described above includes a method usinghigh performance liquid chromatography, in which a chemical-bondedsilica gel including octadecyl, octyl, or phenyl group, a porus-polymergel, or the like is used. The mobile phase includes an aqueous organicwater-soluble solvent, for example, aqueous methanol, aqueousacetonitrile, or the like.

Sphingosine analogues of the present invention represented by the abovegeneral formula (I) are useful as intermediates of syntheses ofTKR1785's or their derivatives. For example, the compound represented bythe above formula (III) is able to be used as an intermediate ofsynthesis of TKR1785-I or its derivative. In order to prepare TKR1785-Ior its derivative from the compound represented by the above formula(III), you can obtain it by synthesizing according to the scheme 1 shownlater.

The compound represented by the above formula (I) is useful to usesyntheses of various sphingolipid analogues as a sphingosine analogouslipid. For example, in order to prepare the compound represented bygeneral formula (V) described below, which has —CH₂OH as X¹ and issimilar to ceramide having a fatty acid such as a palmitic acid bound tothe amino group via a peptide bond, it is needed that a startingmaterial which has an amino group and three hydroxyl groups respectivelyprotected by different protecting groups is used, and is selectivelyremoving the protecting group of amino group, binding a fatty acid via apeptide bond, and removing the protecting group of hydroxyl groups.Additionally, the obtained compound (V) described below can be used tosynthesize a sphingoglycolipid which has a monosaccharide such asgalactose or glucose, or an oligosaccharide at the position of Q⁸ of thecompound shown as the above formula (I). In this case, if Q³ ₁ Q⁵, andQ⁸ are protected by protecting groups which can be removed underdifferent conditions, the protecting group of Q⁸ is selectively removedto allow to use for synthesis of a sphingoglycolipid analogue.

In the formula, Y² is an acyl group of a fatty acid such as myristoyl(C14:0), palmitoyl (C16:0), stearoyl (C18:0), oleoyl (C18:1), orlignoceroyl (C24:0) group.

The sphingosine analogues of the present invention are immunosuppressiveand toxic to tumor cells and have a medical use.

BEST MODE FOR CARRYING OUT THE INVENTION

The following examples illustrate the present invention in furtherdetail but are not intended to define the scope of the invention.

REFERENCE EXAMPLE 1

Synthesis of TKR 1785-1 and TKR 1785-2

From a slant culture of TKR1785 strain (FERM BP-5788), a loop was takento inoculate a 500 ml conical flask containing 100 ml of a liquid medium[Difco yeast nitrogen base 0.67% (w/v) and glucose 2.0% (w/v)] andcultured under shaking at 25° C. for 5 days to provide a seed culture. A1.0 ml portion of this seed culture was inoculated into 18 conicalflasks of 500 ml capacity each containing 125 ml of the above liquidmedium and cultured under shaking (220 rpm) at 25° C. for 9 days. Theresulting culture was centrifuged to separate a supernatant and acellular fraction. The cellular fraction was well mixed and extractedwith 1 L of methanol and the extract was concentrated under reducedpressure. The residue was diluted with 300 ml of water and, aftersufficient mixing, adjusted to pH 2. Then, 300 ml of ethyl acetate wasadded and mixed thoroughly for washing with ethyl acetate. The aqueouslayer was adjusted to pH 9 and extracted with 300 ml of ethyl acetate.The extract was concentrated under reduced pressure to provide 52 mg ofa residue.

This residue was dissolved in 0.4 ml of methanol and subjected to highperformance liquid chromatography to provide two antifungal fractions Iand II. Those active fractions were respectively concentrated underreduced pressure to provide 16 mg of TKR1785-I and 3 mg of TKR1785-IIboth as white powders. The high performance liquid chromatography wascarried out under the following conditions.

Apparatus: LC8A (Shimadzu)

Column: YMCpack C18 (2.0 cm×25 cm) (Y.M.C.)

Mobile phase: 0.05% trifluoroacetic acid-55% (v/v) acetonitrile/water

Physicochemical Properties

JMS-DX302 Mass Spectrometer (Jeol Ltd.) was used for mass spectrometry.JNM-A500 Nuclear Magnetic Resonance Spectrometer (Jeol Ltd.) was usedfor ¹H-NMR spectrometry (in deuterated dimethyl sulfoxide, reference:deuterated dimethyl sulfoxide) and ¹³C-NMR spectrometry (in deuterateddimethyl sulfoxide, reference: deuterated dimethyl sulfoxide). Forultraviolet absorption spectrometry (in methanol), UV-250 self-recordingspectrophotometer (Shimadzu) was used. For infrared absorptionspectrometry (KBr), 270-30 Infrared Spectrophotometer (Hitachi) wasused. L-8500 (Hitachi) was used for amino acid analysis.

The physicochemical properties of TKR1785-I areas follows.

FAB-MS of the purified white powder of fraction-I obtained by highperformance liquid chromatography and concentration under reducedpressure shows m/z 518 [M+H]⁺. Recording of ¹H-NMR and ¹³C-NMR spectraand analysis thereof indicate that this substance has 27 carbon atomsand 3 nitrogen atoms. The ¹H-NMR spectrum and ¹³C-NMR spectrum arepresented in FIG. 3 and FIG. 4, respectively. The ultraviolet adsorptionspectrum of this substance in methanol shows the terminal absorptionsrepresented in FIG. 1. The KBr infrared absorption wave numbers (KBr)are listed below. The IR absorption spectrum of the substance ispresented in FIG. 2.

IR (KBr) (cm⁻¹): 3410, 2920, 2850, 1670, 1540, 1470, 1210, 1140, 1050,840, 800, 720.

The solubility of this substance in various solvents was such that thesubstance is soluble in methanol and water and only sparingly soluble inchloroform and hexane.

The above analytical data revealed that the purified white powderobtained by high performance liquid chromatography and subsequentconcentration of fraction I under reduced pressure is TKR1785-I.Detailed analysis of the ¹H-NMR spectrum presented in FIG. 3 and the¹³C-NMR spectrum presented in FIG. 4 revealed that TKR1785-I has thechemical structure of formula (IIa).

TKR1785-I was subjected to reversed phase partition high performanceliquid chromatography (HPLC) using LC-10A High performance LiquidChromatography System (Shimadzu). The high performance liquidchromatography was carried out under the following conditions.

Column: CAPCELL PACK C₁₈ (6 mm×150 mm) (Shiseido)

Mobile phase: 0.05% trifluoroacetic acid-50% (v/v) acetonitrile/water

Column temperature: 40° C.

Detection UV wavelength: 220 nm

The analysis showed that TKR1785-I is eluted in the position indicatedin FIG. 5.

The physicochemical constants of TKR1785-II are as follows.

The purified white powder obtained by high performance liquidchromatography and concentration of fraction II under reduced pressurewas analyzed for various physicochemical properties. FAB-MS of thissubstance gave m/z532 [M+H]⁺, indicating that it is larger thanTKR1785-I by 14 mass units. This substance was hydrolyzed withhydrochloric acid and analyzed for amino acids. As a result, it wasfound that whereas TKR1785-I contains L-valine, this substance containsL-isoleucine. There was little difference between the UV absorptionspectra of the two substances. The solubility of this substance invarious solvents was also similar to that of TKR1785-I. Analysis of the¹H-NMR spectrum (FIG. 6) and ¹³C-NMR spectrum (FIG. 7) of this substancerevealed that the substance has the chemical structure of formula (IIb).

Based on the above analytical data, the purified white powder obtainedby high performance liquid chromatography and concentration of activefraction II under reduced pressure was found to be TKR1785-II.

TKR1785-II was subjected to HPLC analysis using LC-10A High PerformanceLiquid Chromatography System (Shimadzu). The HPLC conditions were thesame as those used in the analysis of TKR1785-I. The analysis revealedthat TKR1785-II was eluted in the position indicated in FIG. 8.

EXAMPLE 1

Synthesis of Compound (1) from TKR1785-I

To TKR1785-I (10 mg, 19.4 μmol), 6N-HCl (6 ml) wad added and the mixturewas allowed to leave at 110° C. for 15 hours. The reaction mixture wasneutralized by 2N-NaOH (18 ml) under ice-cooling, 6N-NaOH (4 ml) wadadded thereto, and the mixture was stirred for 1 hour under ice-cooling.The reaction mixture was adjusted to pH 6.5 by 2N-HCl under ice-cooling,and concentrated under reduced pressure. Water was added to the residue,and the mixture was extracted with chloroform (total 80 ml). Thechloroform extract was concentrated under reduced pressure, resulting acolorless powder (6.3 mg, 94% yield) of the compound (1) of Table 1.

FAB-MS: m/z 346 (M+H)

Thin layer chromatography (TLC) (chloroform-methanol-acetic acid-water,8:3:1:1): Rf 0.34

UV absorption (in methanol): end absorption

EXAMPLE 2

Synthesis of the Lactone of the Compound (1) from TKR1785-I

To TKR1785-I (20 mg, 38.7 μmol), 6N-HCl (12 ml) wad added and themixture was allowed to leave at 110° C. for 19 hours. The reactionmixture was concentrated under reduced pressure and purified bypreparative thin layer chromatography (TLC) (developed and eluted with asolution of chloroform-methanol-acetic acid, 25:5:1), resulting acolorless powder (5.5 mg) of the lactone of compound (1) of Table 1.

FAB-MS: m/z 328 (M+H)

TLC (chloroform-methanol-acetic acid-water, 8:3:1:1): Rf 0.65

¹H-NMR (DMSO-d₆) δ (ppm): 4.48 (m, 1H), 3.55 (m, 1H), 3.47 (m, 1H), 2.79(dd, 1H), 2.10 (d, 1H), 1.67 (m, 1H), 1.52 (m, 1H), 1.33 (m, 2H), 1.21(s, 22H), 0.84 (t, 3H)

¹³C-NMR (DMSO-d₆) δ (ppm): 176.5, 81.8, 66.7, 50.1, 38.2, 36.2, 31.3,29.1, 29.0, 28.9, 25.1, 22,1, 13.9

EXAMPLE 3

Synthesis of Compound (2) from Compound (1)

Compound (1) (2 mg, 5.8 μmol) was dissolved in dioxane (50 μl), Boc-ON(2.2 mg, 8.7 μmol) and triethylamine (Et₃N) (1.6 μl, 11.6 μmol) wereadded thereto, and the mixture was stirred for 10 hours at roomtemperature. The reaction mixture was concentrated under reducedpressure, 10% aqueous citric acid was added thereto, and extracted withchloroform. The chloroform extract was concentrated under reducedpressure and the residue was purified by preparative TLC (developed andeluted with a solution of chloroform-methanol-acetic acid, 25:5:1),resulting a colorless powder (1 mg) of the compound (2) of Table 1.

FAB-MS: m/z 446 (M+H)

TLC (chloroform-methanol-acetic acid, 25:5:1): Rf 0.59

EXAMPLE 4

Synthesis of Compound (3)

Compound (1) (2 mg, 6.67 μmol) was dissolved in tetrahydrofuran (THF)(5.5 ml), LiAlH₄ (5 mg) was added thereto with stirring underice-cooling. The mixture was treated with reflux for 22 hours, andcooled. Ether (2 ml), water (0.5 ml), and 1N NaOH (0.5 ml) weresucessively added thereto with stirring. The mixture was stirred for 30min, water was added thereto, and extracted with ether (total 40 ml).The ether extract was concentrated under reduced pressure, resulting acolorless powder (2.2 mg) of the compound (3) of Table 1.

FAB-MS: m/z 332 (M+H)

1H-NMR (500 MHz, DMSO-d₆) δ: 7.75 (br), 5.26 (br), 4.77 (br), 4.37 (br),3.74 (br), 3.61 (br), 3.52 (m), 3.02 (m), 1.74 (m), 1.62 (m), 1.50 (d),1.44 (m), 1.35-1.23 (m), 0.84 (t)

EXAMPLE 5

Synthesis of Compound (4)

Compound (3) (10.0 mg, 30.02 μmol) was dissolved in dioxane-water (10:1,300 μl), Boc-ON (11.5 mg, 45.3 μmol) and N,N-diisopropylethylamine(DIEA) (7.9 μl, 45.3 μmol) were added thereto, and the mixture wasstirred for 6 hours. Ethyl acetate was added to the reaction mixture,and the mixture was washed with 10% aqueous citric acid and thensaturated solution of sodium chloride. The ethyl acetate extract wasdried on magnesium sulfate and concentrated under reduced pressure. Theresidue was purified by TLC (developed and eluted with a solution ofchloroform-methanol, 19:1), and compound (4) of Table 1 was obtained asa colorless powder (8.5 mg).

FAB-MS: m/z 432 (M+H)

TLC (chloroform-methanol, 19:1): Rf 0.2

EXAMPLE 6

Synthesis of Compound (5)

Compound (4) (6.5 mg, 15.1 μmol) was dissolved in dichloromethane(CH₂Cl₂) (400 μl), and N,N-dimethylaminopyridine (DMAP) (1 mg), NEt₃(2.5 μl, 18.1 μmol), and t-butyldimethylsilyl chloride (TBDMS-Cl) (2.5mg, 16.6 μmol) were added thereto. The mixture was stirred for 16 hoursat room temperature. The reaction mixture was purified by TLC (developedand eluted with a solution of chloroform-methanol, 100:1) and compound(5) of Table 1 wad obtained as a colorless oil (7.5 mg).

FAB-MS: m/z 546 (M+H)

TLC (chloroform-methanol, 100:1): Rf 0.5

EXAMPLE 7

Synthesis of Compound (6)

Compound (5) (5.0 mg, 9.16 μmol) was dissolved in CH₂Cl₂ (500 μl), andDMAP (2 mg) and Boc₂O (5.9 mg, 27.5 μmol) were added thereto. Themixture was stirred for 18 hours at room temperature. The reactionmixture was purified by TLC (developed and eluted with a solution ofchloroform-methanol, 200:1) and compound (6) of Table 1 wad obtained asa colorless oil (6.0 mg).

FAB-MS: m/z 747 (M+H)

TLC [chloroform-methanol (200:1)]: Rf 0.8

EXAMPLE 8

Synthesis of Compound (7)

To compound (6) (2.5 mg, 3.35 μmol) added THF-acetic acid-water (1:2:1,5 ml), and the mixture was stirred for 17 hours at room temperature. Thereaction mixture was concentrated under reduced pressure and purified byTLC (developed and eluted with a solution of chloroform-methanol, 50:1),resulting a colorless oil (1.8 mg) of compound (7) of Table 1.

FAB-MS: m/z 632 (M+H)

TLC [chloroform-methanol (50:1)]: Rf 0.4

EXAMPLE 9

Synthesis of Compound (8)

Compound (1) (5 mg, 14.5 μmol) was suspended in acetone (900 μl),acetone dimethyl acetal (150 μl) and dl-camphor sulfonic acid (1 mg)were added thereto, and the mixture was stirred for 1 hour at roomtemperature. The reaction mixture was neutralized with NEt₃ (10 μl) andconcentrated under reduced pressure. The residue was purified by TLC[the lower layer of a mixture of chloroform-methanol-water (8:3:1)], andcompound (8) of the Table 1 was obtained as a colorless oil (2.8 mg,yield 51%).

FAB-MS: m/z 386 (M+H)

TLC [the lower layer of chloroform-methanol-water (8:3:1)]: Rf 0.4

EXAMPLE 10

Synthesis of Compound (9)

Compound (8) (1.4 mg, 3.6 μmol) was dissolved in pyridine (100 μl),trichloroethoxycarbonyl chloride (1.5 μl, 10.9 μmol) was added thereto.The mixture was stirred for 30 min under ice-cooling and stirred for 1hour at room temperature. The reaction mixture was purified by TLC (thelower layer of a mixture of chloroform-methanol-water, 8:3:1), andcompound (9) of the Table 1 was obtained as a colorless powder (1.0 mg,yield 42%)

FAB-MS: m/z 734 (M+H)

TLC [the lower layer of chloroform-methanol-water (8:3:1)]: Rf 0.6

TABLE 1

(I) Q⁵ Q⁴ Q³ Q² Q¹ X¹ Compound (1) OH H H H H COOH (2) OH H H H Boc COOH(3) OH H H H H CH₂OH (4) OH H H H Boc CH₂OH (5) OH H H H Boc CH₂OTBDMS(6) OBoc H Boc H Boc CH₂OTBDMS (7) OBoc H Boc H Boc CH₂OH (8) OH Hisopropyl- H COOH idene (9) OH H isopropyl- Troc COOH idene (10)  — — HH H COOH (double bond) (11)  — — H H H CH₂OH (double bond)

REFERENCE EXAMPLE 2

Synthesis of TKR1785-I

TKR1785-I was synthesized according to the method shown in Scheme 1.That is, Boc-NH—CH(CH₂OH)—CH₂OBzl was used as the starting material toprepare HCl.H₂N—CH(CH(CH₃)₂)—CO—NH—CH(CH₂OAc)—CH₂OBzl. This compound(3.0 mg) was coupled with compound (9) (3.0 mg), resulting a protectedderivative of TKR1785-I (3.9 mg). Removal of the protecting group of thecompound by a conventional method gave TKR1785-I as a white powder (1.2mg).

FAB-MS: m/z 518 (M+H)

The final product obtained was analyzed by reversed phase highperformance chromatography (HPLC) to compare with TKR1785-I obtained bypurification of a cultured broth. The condition of HPLC is as follows.

Column: CAPCELL PACK C₁₈ (6 mm×150 mm) (manufactured by Shiseido)

Mobile phase: 50% (v/v) acetonitril/water containing 0.05% trifluoroacetic acid

Column temperature: 40° C.

UV wave-length for detection: 220 nm

As a result, the product obtained by synthesis and the natural productwere eluted at the identical position.

In addition, the final product obtained was dissolved in methanol at theconcentration of 1 mg/ml, tested for the antimicrobial activity againstC. albicans TIMM 0136 using the medium composed of yeast nitrogen base(Difco) 0.67%, glucose 1%, and agar 1.5% by paper disk diffusion method(20 μl/disk of 6 mm in diameter), and showed a prominent growthinhibitory activity.

These results indicated that the compound prepared by the chemicalsynthesis was identical to TKR1785-I. That is, it has found thatcompound (1) is useful as the intermediate for synthesis of TKR1785-I.

TABLE 2 (scheme 1)

INDUSTRIAL APPLICABILITY

The present invention provides a novel lipid, which is useful as anintermediate for syntheses of TKR1785's and their derivatives useful asdrugs for the treatment of fungal infections, allergic diseases, etc.,and also as an intermediate for syntheses of novel sphingolipidderivatives, and a process thereof.

What is claimed is:
 1. A sphingosine analogue represented by thefollowing general formula (I):

In the formula, as for Q¹, Q² and Q³, Q¹ and Q², which are the same ordifferent each other, are hydrogen, alkyl groups having 1-4 of carbonatoms, acyl groups having 2-5 of carbon atoms, or protecting groups ofthe amino group, and Q³ is a hydrogen or a protecting group of thehydroxyl group; or Q² and Q³ make up an isopropylidene group and Q¹ is ahydrogen or a protecting group of the amino group, Q⁴ and Q⁵, which arethe same or different each other, are hydroxyl groups, acyl groupshaving 2-5 of carbon atoms, —O—Q⁶, or hydrogen; or Q⁴ and Q⁵ make up acovalent bond, Q⁶ is a protecting group of the hydroxyl group, X¹ is—COOH, —CONH₂, —CO—Q⁷, —CH₂OH, or —CH₂O—Q⁸, Q⁷ is a protecting group ofthe carboxyl group, and Q⁸ is a protecting group of the hydroxyl group.